Immune complexes may be found in plasma, serum, other body fluids as well as in tissues at sites of putative immune complex deposition. The methods presently available for detection of immune complexes at these various locations are different and often limited to those sites. The invention described herein is exceptionally suitable for the detection of immune complexes found in plasma, serum or other body fluids.
The formation of immune complexes generally results from the interaction of antibodies with antigens and although in most instances, the formation of an immune complex does not lead to evident disease, it often serves a physiological role in homeostasis. See for instance, Theofilopoulos A. N., et al., The Biology and Detection of Immune Complexes, Adv. Immunol. 28:89, 1977. It is, however, important to note that the greatest concentration of immune complexes can be found in patients with infections and autoimmune diseases. Although the comprehension of human immune complex diseases has seen a tremendous expansion within the last twenty years it is to be noted that there is still much physiologic activity related to immune complex formation and resultant effects that requires further clarification, understanding and explanation. To date, the presence of immune complexes has been associated with infections from bacteria, viruses, parasites, different malignancies and with acute and chronic nephritis accompanying serum sickness generally associated with the deposition of immune complex onto tissue. Such human immune complex diseases are often characterized by arthritis and nephritis and may be variably accompanied by leucopenia, agranulocytosis and thrombocytopenia. Further, it has been noted that deposition of immune complexes on blood vessel walls often leads to infiltration of the deposition site by inflammatory cells resulting in the consequential functional impairment of the inflamed organ structure. See for instance: Zubler R., Lambert P., Immune Complexes in Clinical Investigation, Recent Advances in Clinical Immunology, Thompson R.A. (Ed) Churchill Livingstone, N.Y., p. 125, 1977; Carpentier N. A. et al., Clinical Relevance of Circulating Immune Complexes in Human Leukemia, J. Clin. Invest., 60:874, 1977; and Poulton T. A. et al., Immune Complexes in Ovarian Cancer, Lancet 1978.
The term "generalized immune complex disease" has often been used to connote the deposition of circulating immune complexes throughout the body especially likely to occur where conducive factors are present. Typically these include small vessels and slow circulation therethrough. Other considerations such as genetic and host related factors as well as variable tissue affinities influence the rate and extent to which disease systems are produced.
Still other diseases that have been implicated include systemic lupus erythematosus, inflammatory diseases of the bowels generally chronic in nature, glomerulonephritis, and rheumatoid arthritis. See Luthra H. S., et al., Immune Complexes in Sera and Synovial Fluids of Patients with Rheumatoid Arthritis, J. Clin. Invest. 56:458, 1975; and Grigor R., SLE: A Prospective Analysis, Ann. Rheum. Dis. 37:121, 1976; Levinsky R. J., et al., Serum Immune Complexes and Disease Activity in Lupus Nephritis, Lancet 1:564, 1977. The profound local and systemic effects caused by immune complex aggregates in circulation and subsequent deposition as well as their effects on cell mediated immune responses highlights the desirability of detecting circulating immune complexes (CIC) in the clinical environment. A summary and listing of relevant references concerning the effects of immune complexes has been reported in "The Role of Immune Complexes In Disease", WHO Scientific Group, Geneva 1977, ISBN 92 4 120606 3.
Conventionally, immune complexes have been detected by two generic groups of methods, namely the physical-chemical methods which evaluate particular characteristics of complex size, charge and temperature dependent solubility while the biological assay methods are generally based on immune complex effects on receptor systems such as blood cells and platelets, or on soluble protein such as complement-N-rheumatoid factor. Of these two groups, it is the latter in which this invention may be most appropriately associated.
Biological methods directed against humoral receptors include inactivation of CH.sub.50 units in reference serum, inhibition of .sup.125 I-Clq binding, insolubility of reaction product between Clq in complexes, fixation of .sup.125 I-Clq to the Fc fragments of complexed Ig, binding of Fc in complexes to insolubilized Clq, fixation of RhF and Clq to the Fc portion of complexed antibodies, and affinity of conglutinin for complex bound C3. Methods directed against cellular receptors include interaction with platelet Fc receptors as well as interaction with C3b receptors on a lymphoblastoid cell line such as a Raji cell. These methods have been generally referenced and described in Nydeggar V. E., Soluble Immune Complexes In Human Disease, CRC Critical Review in Clinical Laboratory Science, July 1980, pages 123-170; Theofilopoulos A. N. et al., Detection of Immune Complexes: Techniques and Implications, Hospital Practice, May 1980; and Zubler R., et al., Detection of Immune Complexes in Unheated Sera by a Modified I-Clq Binding Test, J. Immunol. 116(1):232, 1976.
The foregoing list, although representative, is still not comprehensive as additional methods have been described and compared for effectiveness in specific disease conditions in "A WHO Collaborative Study For the Evaluation of 18 Methods For Detecting Immune Complexes And Serum" by Lambert et al., J. Clin. Lab. Immuno. 1:1-15, 1978.
Included in that report are five methods directed towards the detection of Clq, a complement protein. One method relies on the principle that Clq acts as a receptor for aggregates containing either IgG or IgM, and further involving rheumatoid factor dependent on the affinity of IgMrf for aggregates of IgG or IgA. Another method is directed to conglutinin binding which is, in turn, dependent upon a Ca++ dependent interaction of bovine conglutinin with immune complex bound C3b that has been cleaved by a C3b inactivator. Also included are methods directed gainst receptors of C3b or C3d with complement coated immune complexes operative in Raji cell radioimmune type assays; C3b or C3d lymphocyte rosette formation inhibition tests; and the interaction of immune complexes with the Fc portion of immunoglobulins in the formats of: platelet aggregation, macrophage uptake inhibition, K cell cytotoxicity inhibition, and neutrophil inhibition tests.
These methods and tests for the detection of immune complexes have traditionally suffered from their overall complexity and their extensive, critical reagent requirements. It is an object of the present invention to provide a test kit utilizing greatly simplified procedure especially useful in clinical environments where efficiency and throughput or turnover time are of paramount importance.
A test involving rheumatoid factor and Clq for the analysis of antigen antibody complexes has been described in U.S. Pat. No. 4,143,124 to Masson et al. Although this reference is primarily directed towards the detection and analysis of antigen antibody formation, it relief on the fact that such a formation has the ability to combine with rheumatoid factors as well as the Clq components of complement. Entailed in the disclosed test is the insolubilization of rheumatoid factor or Clq onto a solid-phase substrate which is then contacted with a fluid containing the immune complexes to be detected. The reacted and immobilized immune complexes are then freed from the substrate and allowed to react with soluble rheumatoid factor or Clq and particles such as red blood cells coated with immunoglobulins. Agglutination of the particles is interpreted to indicate the absence of immune complexes in the original sample. Such a procedure disadvantageously entails procedural steps for freeing the bound immune complexes to permit subsequent reaction therewith. Further, the test depends upon the complete lack of cross-reactivity of rheumatoid factor or Clq with free antigen or free antibody in the original sample fluid.
It is an object of the present invention to avoid these disadvantages and to provide a simplified system for immune complex detection in kit form.
Another assay procedure for immune complexes is described by Soothill et al in U.S. Pat. No. 4,141,965. A kit, utilizing the same assay procedure, has been provided in Soothill's U.S. Pat. No. 4,210,622. The procedure taught analyzes immune complexes by detecting a constituent thereof. In order for the system to be effective, the constituent must be able to produce non-human IgM antibodies and, to be attachable to a latex particle. The constituent, after attachment to the latex particle, and after addition to IgM and to the immune complex containing sample, permits detection of immune complexes as indicated by the absence of agglutination of the latex particles.
It is an object of the present invention to provide a kit employing a system that is not limited to the production of non-human IgM antibodies nor requiring the attachment of such a constituent to a latex particle.